Thymocyte-stimulating factor from peripheral blood cells.

Human peripheral blood leukocytes (HPBL) produce a thymocyte-stimulating factor (TSF-HPBL) that enhances the phytohemagglutinin (PHA) and concanavalin A (Con A) responsiveness of murine thymocytes. This activity is considerably specific for thymocytes. TSF-HPBL is not mitogenic by itself. Experiments with cell cultures pretreated with carbonyl iron particles showed that phagocytic cells are not involved in the production of mouse and rat TSF but are involved in the production of TSF-HPBL. The dose-response profile to PHA of murine thymocytes cultured in the presence of TSF-containing supernatants is similar to that of mature, immunocompetent spleen cells. TSF-HPBL, however, does not enhance the PHA responsiveness of murine thymocytes at low (<0.25 μg/microwell) concentrations of mitogen. TSF enhances the PHA and Con A responsiveness of the high-density subpopulations of thymocytes isolated on a Ficoll-Hypaque gradient. In general, the enhancing effect of TSF-HPBL on these subpopulations of thymocytes is smaller than that exerted by TSF. While supernatants containing TSF confer to thymocytes the ability to participate in a mixed lymphocyte reaction, this effect is not exerted by supernatants containing TSF-HPBL. A factor enhancing the PHA and Con A responsiveness of murine thymocytes is also produced by murine peripheral blood leukocytes (TSF-MPBL). This factor, similarly to TSF-HPBL, is produced by phagocytic cells and does not confer to murine thymocytes the ability to participate in a mixed lymphocyte reaction. Human T-cell lines do not enhance the PHA or Con A responsiveness of murine thymocytes. TSF-HPBL has a molecular weight of about 30,000 daltons, as measured by Sephadex filtration. Its half-time of inactivation as 56 °C is 162 ± 8 min.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

DNA synthesis by cultured lymphocytes: a modified method for measuring 3H-thymidine incorporation

A rapid, convenient and inexpensive method for harvesting lymphocyte cultures and measuring the incorporation of ³H-thymidine into trichloroacetic acid precipitable material has been developed. The basic principle is to adsorb the entire contents of a microculture well onto the cotton applicator portion of a Q-tip, precipitate the DNA, wash away unincorporated ³H-thymidine, and count the remaining ³H in a mixture of scintillation fluid plus detergent. Data presented for mixed lymphocyte cultures between allogeneic rat lymph node cells, mixed lymphocyte cultures of human peripheral blood lymphocytes, Con A stimulated mouse spleen cells, and PHA stimulated mouse spleen cells show the method to be highly reproducible with standard deviations of less than 15% of the mean for quadruplicate mixed lymphocyte cultures and in most cases less than 5% of the mean for duplicate mitogen stimulated cultures. This culture system also gives positive values for PHA stimulated DNA synthetic responses of mouse spleen cells cultured in RPMI-1640 plus penicillin and streptomycin but without exogenous serum.     

Some effects thymocyte-stimulating factor.

Thymocyte-stimulating factor (TSF) produced in supernatants of murine spleen cells stimulated with mitogens or with allogeneic cells confers to thymocytes the ability to respond to concanavalin A (Con A) with the dose-response characteristic of mature, immunocompetent T cells. No enhancement of the responsiveness of bone marrow cells to lipopolysaccharide (LPS) was found. Thymocytes from mice of different strains acquire, after treatment with TSF, a responsiveness to phytohemagglutinin (PHA) and Con A proportional to that shown by spleen or lymph node cells from mice of the same strain. It was also shown that murine thymocytes treated with TSF cause a graft-vs-host reaction when injected into an appropriate hybrid. All these activities are thermolabile and disappear from supernatants at the same rate, thus showing that they are, very probably, due to the same substance. Spleen cells of mice bearing tumors causing splenomegaly (C3HBA and H2712 adenocarcinomas) show a decreased production of TSF if judged on the basis of TSF produced per million spleen cells. Rat spleen cells produce a substance (rat TSF) which stimulates the PHA and Con A responsiveness of murine thymocytes. Rat TSF has a molecular weight similar or identical to that of murine TSF. However, on the basis of the different rates of thermodenaturation, it appears that rat TSF and murine TSF are two different molecules.     

Production of thymocyte-stimulating factor by murine spleen lymphocytes: cellular and biochemical mechanisms.

Cultures of murine spleen lymphocytes treated with Thy 1.2 antiserum plus complement do not produce thymocyte-stimulating factor (TSF). The population of thymocytes composed of immunocompetent, low-density cells produces only small amounts of TSF. Experiments with cyclophosphamide-injected mice and with spleen cells treated in vitro with antiserum to the murine B lymphocyte antigen plus complement and experiments using spleen cells stimulated in vitro with Sepharose-bound phytohemagglutinin indicate that B lymphocytes neither cooperate with T lymphocytes for the production of TSF nor produce TSF. Some lectins (pokeweed mitogen, Lens culinaris hemagglutinins A and B) have been found to induce the production of TSF by spleen cells. Other lectins (wheat germ agglutinin, Agaricus bisporus agglutinin) and sodium periodate do not. Spleen cells of mice immunized in vivo with keyhole limpet hemocyanin bound to bentonite particles or with BCG produce TSF when challenged in vitro with the specific antigen. Experiments using inhibitors of the macromolecular metabolism showed that DNA synthesis is not required for the production of TSF by spleen lymphocytes, whereas RNA and protein synthesis are required. Resolution of spleen lymphocytes on a discontinuous albumin gradient into six subpopulations showed that the TSF activity was rather uniformly distributed among the various subpopulations of cells.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Specific activation of murine B cells by low molecular weight polyamino-polycarboxylic acids (ampholytes).

Commercial preparations of ampholytes (AM), which consist of mixtures of large numbers of polyamino-polycarboxylic acids of molecular weight less than 1000 and whose chemical composition is otherwise not specified by the manufacturer, were found to induce blastogenesis in spleen cell cultures from various strains of mice. The blastogenic response of the spleen cells to the ampholytes was 2 to 12 times greater than that of the unstimulated cultures, and peak stimulatory activity occurred 2–4 days after stimulation. Preparations consisting of either acidic, neutral, or alkaline ampholytes were all found to be mitogenic, although the alkaline ampholytes generally induced the highest stimulation and were active over the widest concentration range (0.08–80 μg per culture). Studies using spleen cells from nu/nu mice, CBA/N mice, organ distribution studies, and the use of cytotoxic antisera and complement for the depletion of thymus-derived (T) cells or bone marrow-derived (B) cells suggested that ampholytes are mitogenic for murine B cells. These cells arise early in ontogeny, because the ampholytes were found to be as mitogenic for spleen cells from newborn as for adult mice. Further, in concordance with the characteristics of other B-cell mitogens, injection of mice with ampholytes induced polyclonal antibody synthesis. The possibility that blastogenic stimulation was due to a contaminant was ruled out by demonstrating that anti-lipid A-treated, ultrafiltered, and isoelectric-focused ampholytes retained stimulatory activity. The results of these investigations suggest that commercial ampholyte preparations contain an undetermined number of low molecular weight (< 1000) acidic, neutral, and alkaline polyamino-polycarboxylic acids which are specific mitogens for a primitive population of murine B cells.     

Paradoxical production of mouse thymocyte activating factor by ouabain-treated human mononuclear cells

At concentrations as low as 10(-7) M, the cardiotonic glycosteroid ouabain, a specific inhibitor of the membrane Na+, K+-ATPase, is known to inhibit in vitro human lymphocyte proliferation produced in mixed lymphocyte cultures or induced by various stimulating agents (PHA, Con A, PWM, soluble antigens), while mouse lymphocyte proliferation is unaffected at this concentration. Ouabain inhibits most of proliferative response parameters at all stages of the transformation. This observation prompted us to suggest that ouabain could also act through inhibition of interleukin production which is known to occur during the first hours after T-cell stimulation in the presence of monocytes. In order to check the possible influence of ouabain on interleukin production, conditioned media from stimulated human mononuclear cells, prepared in the presence or in the absence of inhibitor, were tested for their ability to promote a mouse thymocyte response to PHA. Instead of the expected inhibition, we found that ouabain, even at high concentrations (2 X 10(-6) M) enhanced the stimulatory effect and/or the production of murine thymocyte activating factor(s). Moreover conditioned media from serum-free cultures of unstimulated human mononuclear cells exposed for 24 hr to low ouabain concentrations (10(-8) to 10(-7) M) showed a high activating effect on the response of murine thymocytes to PHA. This soluble factor produced upon ouabain treatment is produced by adherent cells and appears to be functionally similar to interleukin 1.     

Effects of a thymic hormone,THF, on tumor-bearing mice and tumor-resected mice

Summary The administration of THF (thymus humoral factor) to mice bearing a chemically induced fibrosarcoma was followed by a significant improvement of the in vivo anti-tumor reactivity, as measured in the Winn assay, and of the in vitro response to PHA (phytohemagglutinin) of spleen cells. When THF treatment was given to mice after local resection of various metastasizing tumors, the subsequent death rate and survival time were not different from those in controls, and the anti-tumor reactivity of spleen cells of these THF-treated tumor-resected mice was not modified. Moreover, in contrast to tumor-bearing mice, a reduction in the PHA response of spleen cells from tumor-resected mice was noticed. The relevance of the immunologic status before THF treatment is discussed with reference to the present findings.Abbreviations THF thymus humoral factor - PHA phytohemagglutinin - cpm counts per minute - GvH graft-vs-host - MLC mixed lymphocyte culture - BSA bovine serum albumine - FS fibrosarcoma - SE standard error - SD standard deviation The Harold L. Korda Professor of Cancer Research     

In vitro viability of lymphoid cells from lines of mice genetically selected for high or low responsiveness to phytohemagglutinin

The kinetics of viability of lymph node and spleen cells of mice genetically selected for "high" or "low" in vitro lymphocyte responsiveness to PHA were studied in PHA or PPD-stimulated short-term cultures. Lo/PHA cells were found to be less viable than Hi/PHA cells in unstimulated control cultures. PHA improved the viability of Lo/PHA cells while inducing proliferation of Hi/PHA cells with the appearance of more and larger lymphoblasts in the latter. PPD only improved the viability of spleen cell cultures, more so for the Hi/PHA line. The interline difference in thymidine uptake was smaller after PPD than after PHA stimulation. Modifications of culture conditions designed to decrease the interline difference in cell viability lessened but did not abolish the separation between the two lines for the PHA response as measured by thymidine uptake.     

Biochemical characterization of lymphocyte regulatory molecules. II. Purification of a class of rat and human lymphokines

Rat spleen cells activated in vitro by concanavalin A produce lymphokine molecules that possess biologic activity in a number of murine lymphocyte response assays. A single class of lymphokine most adequately described as T cell growth factor (TCGF, Interleukin-2) with a m.w. of 15,000 as estimated from gel filtration studies and with an isoelectric range of 5.4 to 5.6 stimulates i) the growth of established T cell lines in culture, ii) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is non-mitogenic, iii) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) mouse spleen cell cultures, and iv) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures and in nude mouse spleen cell cultures. We suggest that in each of the assay systems tested, this class of rat lymphokine acts directly on activated T cells. Nonactivated T cells must be stimulated by either mitogen or antigen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued lymphokine-dependent proliferation. Similarly, human peripheral blood mononuclear cells activated by phytohemagglutinin (PHA) produce a class of lymphokines of identical size with an isoelectric point of 6.0 to 6.5 that possess the same biologic properties as measured in murine lymphocyte response systems.     

Effects of lymphocytes from Marek's disease-infected chickens on mitogen responses of syngeneic normal chicken spleen cells.

PHA responses have been measured in lymphoid cell cultures prepared by mixing normal chicken spleen cells with spleen or thymus cells from syngeneic chickens infected with the oncogenic herpesvirus MDV. Results of these studies may be summarized as follows: 1) spleen cells from MDV-infected chickens with visceral lymphomas inhibit the PHA response of normal spleen cells possibly by release of soluble inhibitory factors in response to the mitogen; 2) lymphoid cells from asymptomatic MDV-infected chickens, although hyporeactive themselves to PHA, can have a stimulatory effect on PHA responses of normal spleen cells in mixed cultures; 3) spleen cells from MDV-infected chickens, effectively protected from viral oncogenesis by HVT vaccination, show normal reactivity to PHA in spearate cultures and may react in mixed cultures like normal lymphocytes, with neither a pronounced stimulatory nor inhibitory effect on the PHA response of normal spleen cells.     

Mastocytoma-medicated suppression of mixed lymphocyte culture and mitogen responsiveness.

Murine spleen cells, stimulated in vitro by allogeneic spleen, display a strong proliferative response with the subsequent development of cytotoxic cells. This proliferation and sensitization can be abrogated by the addition of mitomycin-treated or X-irradiated murine DBA/2 mastocytoma cells (P-815). The substance required for this depression of lymphocyte responsiveness is present in the cell-free supernatant fluids of P-815 cultures. The suppression appears to be due to interference with cell proliferation in the mixed lymphocyte culture, because the P-815 also prevents spleen cells from proliferating in response to the mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and phytohemagglutinin (PHA). The significance of these findings is discussed.     

Suppression of mitogen- and tumor-cell-induced lymphocyte stimulation by tumor-associated fetal antigens

The ability of tumor-associated fetal antigens (TAFA) to suppress mitogen and tumor-cell-induced blastogenesis was investigated. Three different TAFA (I, III, and IV) were tested in PHA and Con A lymphocyte proliferation assays. Spleen cells from New Zealand black rats (NBR) were fractionated over nylon wool; and nonadherent (NA) and adherent (AD) cells were compared with unfractionated (UF) cell responses. Preincubation of NA cells with TAFA-I was followed by addition of phytohemagglutinin (PHA) elicited suppression in a 3-to 4-day assay. UF cells were unresponsive to TAFA and/or PHA at all concentrations tested. TAFA dose—response titration curves in Con A proliferation assays revealed that all TAFA tested (TAFA I, -III, and -IV from fibrosarcomas; TAFA-I and -III from osteosarcomas) were suppressive. For some TAFA, nanogram quantities produced significant suppression. In mixed leukocyte tumor cell assays (MLTC) both UF and NA normal rat spleen cells were tested for proliferative responses to syngeneic tumor cells. Four distinct TAFA, purified by high-pressure liquid chromatography, suppressed lymphocyte proliferation when added to MLTC cultures in 5-day assays. Specificity experiments demonstrated that trinitrophenol-bovine serum albumin did not produce similar immunosuppression. TAFA did not block recognition of tumor antigen nor produce nonspecific cytotoxicity of the spleen cells. Significant suppression of DNA synthesis was produced by TAFA-1 following cocultivation with spleen and tumor cells for 1, 2, and 3 days, compared to no suppression when spleen and tumor cells were washed free of TAFA-I prior to tumor cell addition at Day 0. Similar experiments using rat embryo fibroblasts (REF) as stimulators demonstrated that pre-REF cocultivation treatment of lymphocytes with TAFA-I significantly reduced subsequent blastogenic responses. This effect was not reversible; however, if TAFA-I was added to responders previously stimulated by REF, a suppressive response was not seen. Experiments were also carried out to determine the reversibility of TAFA-I effects. Cells were treated with TAFA-I from 1 to 5 days, followed by determination of lymphocyte blastogenesis. TAFA-I effects are reversible and antigen presence is required to completely suppress (or inhibit) stimulation by tumor cells.     

Enhanced uptake of calcium by transforming lymphocytes

Phytohemagglutinin caused a rapid increase in calcium accumulation by lymphocytes. The enhanced uptake was observed within 1 hr of initiation of transformation in both human lymphocyte and mouse spleen cell cultures. Increased uptake was also found in mixed lymphocyte cultures although not until late in the response. The rate of calcium uptake increased with time after stimulation and depended upon the PHA concentration. The lowtemperature coefficient (Q₁₀) for calcium permeability in unstimulated cells was indicative of a passive diffusion process, but the Q₁₀ was slightly greater for PHA-stimulated cells. Various chemical agents which alter membrane properties and/or cellular metabolism inhibited uptake to a greater extent in stimulated cultures than in control cultures. Ouabain did not affect the calcium permeability of controls or stimulated cells within 1 hr after PHA addition, but it partially inhibited calcium uptake 12 hr after PHA treatment. Cyclic AMP, dibutyryl cyclic AMP, and theophylline also altered calcium transport providing evidence for an effect of cyclic AMP on an early event in the transformation process.     

Mechanism for Macrophage Activation against Corynebacterium parvum—Participation of T Cells and Its Lymphokines

It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T-cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c- + / + mice was abrogated completely by the pre-treatment of spleen cells with anti-Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c-nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin-P (a PHA-induced lymphokine) made it possible for BALB/c-nu/nu mice to produce TNF, associated with an induction of Lyt-1⁺ cells and Lyt-2⁺ cells. However, treatment with the lymphokine did not augment the increases of Ia⁺ cells or liver and spleen weights. These results suggest that increasing the number of Ia⁺ cells is not sufficient to bring about TNF production; Ia⁺ cells must also be stimulated by T cells or T-cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia⁺ cells against C. parvum.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Effects of costimulator on immune responses in vitro.

We recently described a factor, costimulator, that is required for the concanavalin A-induced proliferation of CBA mouse thymocytes in vitro (see Reference 1). Using the costimulator dependence of mouse thymocytes as an assay, we have now determined that spleen cells from congenitally athymic (nude) BALB/c mice do not produce costimulator in response to Con A, and spleen cells depleted of Thy 1-positive cells do not respond to it in the presence of Con A. Thus, costimulator both requires thymus-derived (Thy 1+ lymphocytes for its production and has an effect on this type of cell. (However, the costimulator-producing and responsive cells may be different.) Purified costimulator preparations are a source of the required second component for the stimulation of adult, CBA/J thymic lymphocytes by PHA, normally a poor mitogen for these cells. They also enhance the level of DNA synthesis in a mixed leukocyte reaction, and the specific generation of cytotoxic lymphocytes to allogeneic tumor cells in vitro. Costimulator is not H-2 restricted in its effects, and it is produced in mixed leukocyte reactions. Finally, it has been possible to grow normal, primary thymic lymphocytes in culture for about 20 days by adding partially purified costimulator to the cultures.